1. Field of the Invention
The present invention relates to new means for detecting and analysing the interactions between proteins
2. Discussion of the Related Art
Protein/protein interactions are fundamental cellular mechanisms which are involved in the formation of multimeric complexes responsible both for functions such as transcription and translation, and for the transmission of signals, the response to pathogenic agents and the like.
To analyse these interactions, conventional biochemical techniques (cross-linking, co-immunoprecipitation, co-fractionation by chromatography) which are intended to isolate the proteins interacting with a target protein are generally difficult to use, especially when the interacting proteins are in a small quantity. In addition, they make it possible to identify the interacting proteins, but not to directly obtain the genes corresponding to the said proteins.
One procedure called: "double-hybrid method", which makes it possible to detect interactions between 2 proteins, has been developed by FIELDS and SONG FIELDS and SONG, Nature, 340, 245-246 (1989) and Patent U.S. Pat. No. 5,283,173!.
This procedure is based on the co-expression, in the same yeast cell, of the following genes:
one reporter gene expressing a detectable protein, whose level of expression depends on transcriptional activation by a polypeptide domain; and
two chimeric genes, encoding two hybrid proteins comprising respectively the sequences of the two proteins whose interaction it is desired to detect: one comprises, in addition, a transcription activation domain regulating the expression of the reporter gene, the other comprises, in addition, a DNA-binding domain, which recognizes a binding site situated on the reporter gene in the host cell.
When the two chimeric genes are expressed in the same cell, if an interaction occurs between the two proteins, it causes the transcription activation domain and the reporter gene to come into contact due to the attachment of the DNA-binding domain to its site situated on the reporter gene. The transcription of the latter is activated, and an increase in its expression product can therefore be observed.
In the system described in Patent U.S. Pat. No. 5,283,173, FIELDS and SONG exploited more particularly the properties of GAL4, a transcription activator in Saccharomyces cerevisiae.
GAL4 activates the transcription by RNA polymerase II (PolII) of genes encoding enzymes involved in the metabolism of galactose. This protein comprises 2 functionally independent and physically separable domains: one DNA-binding domain, represented by the N-terminal domain (amino acids 1-147) which binds to specific sequences of the DNA (UAS.sub.G, for Upstream Activating Sequence for Galactose), and a transcription activation domain represented by the C-terminal domain (amino acids 768-881), which activates the transcription by PolII.
Two types of hybrid proteins can thus be produced from GAL4: one contains the GAL4(1-147) domain fused to a first test protein, the other contains the GAL4(768-881) domain fused to a second test protein. If the 2 test proteins are capable of interacting, they bring the two domains of GAL4 closer and trigger the transcription of the reporter gene (for example the lacZ gene encoding the .beta.-galactosidase of E. coli).
It is possible to simultaneously test several proteins with the aim of investigating their interactions with a given protein. For example, the protein whose partners are sought is fused with the binding domain GAL4(1-147) and it is tested against a library of proteins fused with the activator domain GAL4(768-881).
Numerous improvements have been made to the technique initially used by FIELDS and SONG: for example a system for cloning into .lambda. phages subsequently convertible to plasmids by recombination of lox sites, has been developed; a second reporter gene, consisting of a marker for auxotrophy, the HIS3 gene (involved in the metabolism of histidine), was used in combination with the lacZ gene in order to eliminate the false positives more efficiently DURFEE et al., Genes & Development, 7, 555-569, (1993)!.
Variants of this technique have also been proposed: thus the non-exclusive aspect of 2 interactions of a protein A with 2 other proteins B and C can be demonstrated by the difference in transcription activation between a system comprising only the 2 hybrids B and C fused with GAL4-(1-147) and GAL4-(758-881) respectively, and a system comprising the 2 hybrids B and C as well as the protein A non-fused but simply overexpressed, and which forms an intermediate between B and C LEGRAIN and CHAPON, SCIENCE, 262, 108-110, (1993)!.
A derived technique, called single-hybrid system, makes it possible to detect proteins attaching to DNA fragments (for example promoters or fragments involved in the replication of the DNA). In this case, only the hybrid proteins encoded by a library of cDNAs fused with GAL4-(768-881) are used. This library is introduced into cells containing a reporter gene (HIS3 or lacZ) placed downstream of the DNA fragment studied WANG and REED, NATURE, 364, 121-126, (1993); LI and HERSKOWITZ, SCIENCE, 262, 1870-1874, (1993)!.
Related strategies have also been developed for identifying the DNA sequences to which a given protein binds WILSON et al., SCIENCE, 252, 1296-1300, (1991)!, or for selecting functional mutations in protein domains which bind to DNA WILSON et al., Proc. Natl. Acad. Sci. USA, 90, 9186-9190, (1993)!.
The major limitation of this system is linked to the fact that the protein whose partners are being sought should not exhibit PolII transcription activating activity. This limit therefore excludes the study, by this technique, of the physiological PolII activators (involved in pathological or normal PolII transcription regulation mechanisms), and, in general, of the proteins which, fortuitously, possess activating properties. Now, such properties are not rare: PolII transcription activation by proteins which are not physiological PolII activators has been reported; it has also been shown that 1% of the E. coli genomic fragments randomly generated by digestion with Sau3A, when they are fused with the domain encoding GAL4(1-147), encode peptides which activate the PolII transcription MA and PTASHNE, Cell., 51, 113-119, (1987)!.
The inventors have undertaken the development of a system which does not possess this limitation. For this purpose, they had the idea of searching for a method based on the use of the PolIII system.
However, to achieve a double-hybrid system based on the use of polymerase III, it was necessary, on the one hand, to identify transcription factors for the PolIII system capable of being used for this purpose, and, on the other hand, to have an appropriate reporter gene.
The PolIII system transcribes "housekeeping" genes, whose products (tRNA, 5S rRNA, U6 RNA and the like) are necessary for the basic functions (translation, splicing and the like) of any active cell. The functioning of the PolIII system requires the presence of various transcription factors which ensure correct positioning of polymerase III. For example, the transcription of the tRNA genes involves, first of all, a factor called TFIIIC or .tau. which binds to intragene sequences called A block and B block. The attachment of .tau. onto the B block involves the subunit .tau.138 and its attachment to the A block, the subunit .tau.95. Once attached, .tau. then allows the recruitment of another factor, called TFIIIB, within the vicinity of the site of initiation of transcription. For its part, TFIIIB allows the recruitment and positioning of polymerase III on the gene.
In the case of the gene for U6 RNA (called SNR6), the PolIII promoter comprises an intragene A block, a B block downstream of the signal for termination of transcription of the gene, and a TATA box at position -30. A succession of steps analogous to that described for the tRNA genes makes it possible to initiate the transcription of SNR6. These steps are schematically represented in FIG. 1. The .tau.138 subunit of the .tau. factor first binds to the B block (1), then the .tau.95 subunit to the A block (2). The attachment of .tau. makes it possible to recruit TFIIIB via the .tau.131 subunit (3). The precise role of the other subunits (.tau.50, .tau.60 and .tau.91) has not yet been established. TFIIIB, which is composed of subunits of 70 kD (70), 90 kD (90) and of TBP (TATA-binding protein, a protein attaching to the TATA box) is positioned upstream of SNR6 (4) and then allows the recruitment of the polymerase PolIII (5). The binding of .tau.138 onto the B block of SNR6 therefore constitutes one of the first stages of the transcriptional activation of this gene. A mutation of the B block (deletion of bases +238 and +239 relative to the site of initiation of transcription of SNR6) which abolishes this binding prevents the transcription of the SNR6 gene BROW and GUTHRIE, Gene & Development 4, 1345-1356 (1990); BURNOL et al., Nature, 362, 475-477, (1993)!.
During previous studies, the inventors demonstrated that it was possible to re-establish the transcription of the SNR6 gene by inserting, inside the mutated B block of SNR6, UAS.sub.G sequences, and by using a chimeric transcription factor, GAL4-(1-147)-.tau.138, resulting from the fusion of the binding domain GAL4-(1-147) and .tau.138 MARSOLIER et al., Proc. Natl. Acad. Sci. USA, 91, 11938-11942, (1994)!. This transcription mechanism is schematically represented in FIG. 2. These experiments were carried out in the presence of a wild-type SNR6 gene, in order to produce transcripts in a quantity sufficient to ensure cell viability.